Electronic Journal of Biotechnology ISSN: 0717-3458 http://www.ejbiotechnology.info DOI: 10.2225/vol16-issue5-fulltext-14 RESEARCH ARTICLE The bacterial community structure in an alkaline saline soil spiked with anthracene Carolina Castro-Silva1 · Víctor M. Ruíz-Valdiviezo1 · César Valenzuela-Encinas2 · Rocio J. Alcántara- Hernández3 · Yendi E. Navarro-Noya1 · Edgar Vázquez-Núñez1 · Marco Luna-Guido1 · Rodolfo Marsch1 · Luc Dendooven1 1 Centro de Investigación y de Estudios Avanzados, Departamento de Biotecnología y Bioingeniería, Laboratorio de Ecología de Suelos, México 2 Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, México 3 Universidad Nacional Autónoma de México, Instituto de Ecología, Laboratorio de Ecología Bacteriana y Epigenética Molecular, México Corresponding author: [email protected] Received January 18, 2013 / Accepted September 12, 2013 Published online: September 15, 2013 © 2013 by Pontificia Universidad Católica de Valparaíso, Chile Abstract Background: The application of polycyclic aromatic hydrocarbons (PAHs) will affect the bacterial community structure as some groups will be favoured and others not. An alkaline saline soil with electrolytic conductivity (EC) 56 dS m-1 was spiked with anthracene and acetone while their effect on bacterial community structure was investigated. Results: The percentages of Acidobacteria and Actinobacteria decreased over time, while the percentage of Proteobacteria, mostly Xanthomonadales, increased. The percentage of the phylotypes belonging to the Nocardioides, Rhodococcus and Streptomyces, known degraders of PAHs, was larger in the anthracene-amended soil than in the acetone-amended and unamended soil at day 14. The phylotypes belonging to the genera Sphingomonas, also a known degrader of PAHs, however, was lower. Weighted and unweighted PCoA with UniFrac indicated that phylotypes were similar in the different treatments at day 0, but changed at day 1. After 14 days, phylotypes in the unamended and acetone-amended soil were similar, but different from those in the anthracene-spiked soil. Conclusions: It was found that incubating the soil and contaminating it with anthracene changed the bacterial community structure, but spiking the soil with acetone had little or no effect on the bacterial community structure compared to the unamended soil. Keywords: C dynamics, N mineralization, phylogenetic analysis, polycyclic aromatic hydrocarbons, principal component analysis, UniFrac. INTRODUCTION Mexico is an important petroleum producing country so contamination during extraction and transport occurs frequently. In Mexico, 27971 ton oil contaminated the environment as a result of leaks and spills in 2010. Polycyclic aromatic hydrocarbons (PAHs) are important components of petroleum. They are resistant to degradation and have been listed as priority pollutants by both the US Environmental Protection Agency and European Union (Doyle et al. 2008). DOI: 10.2225/vol16-issue5-fulltext-14 1 Castro-Silva et al. Consequently PAHs, such as anthracene, have often been used as a model in the study of factors controlling the removal of hydrocarbons from soil (Vázquez-Núñez et al. 2009; Zhang et al. 2011b). Anthracene is a tricyclic aromatic hydrocarbon and has been found in fumes from vehicle exhaust, coal, coal tar, tobacco smoke and at waste sites. Humans exposed to anthracene experienced headaches, nausea, loss of appetite, inflammation or swelling of the stomach and intestines (ATSDR, 1995). Microorganisms remove PAHs from soil and even complex compounds, such as anthracene with three benzene rings, are dissipated within weeks (Amezcua-Allieri et al. 2012). Numerous bacteria have been described that can degrade PAHs, such as phylotypes belonging to the genus Sphingomonas (Jurelevicius et al. 2012), Achromobacter (Tiwari et al. 2010), Acidovorax (Jurelevicius et al. 2012), Herbaspirillum (Louvel et al. 2011), Methylibium (Zhang et al. 2012), Polaromonas (Jurelevicius et al. 2012) and Variovorax (Zhang et al. 2011a), so they will be favoured (Jones et al. 2011). Contaminating soil with PAHs will also inhibit certain microorganisms (Wang et al. 2011). As such, changes in the bacterial community structure will be indicative of the effect of anthracene. An alkaline saline soil of the former lake Texcoco (Mexico) with electrolytic conductivity (EC) 56 dS m-1 and pH 9 was spiked with acetone, contaminated with anthracene dissolved in acetone or left unamended. The C and N dynamics, and anthracene concentrations were monitored in an aerobic incubation while the bacterial community structure was determined after 0, 1 and 14 days. The objective of this study was to investigate how incubation time, acetone and anthracene affected the bacterial community structure in an alkaline saline soil. MATERIALS AND METHODS Site description and soil sampling The sampling site is located in the former lake Texcoco in the valley of Mexico City. The soil was sampled at random by augering the 0-10 cm layer of two 0.5 ha plots. The soil from each plot was pooled so that two soil samples were obtained (n = 2). This field-based replication was maintained in -1 -1 -1 the incubation study. The pHw in the sandy soil (clay 22 g kg , silt 106 g kg and sand 872 g kg ) was 9.0, electrolytic conductivity 56 dS m-1, water holding capacity (WHC) 504 g kg-1 soil and organic carbon content 12.9 g kg-1 soil. Techniques used to characterize the soil are described in Fernández- Luqueño et al. (2008). Treatments and aerobic incubation Four different treatments were applied to the two soil samples (n = 2). First, 20 g sub-samples were amended with 2 mL acetone (acetone-amended soil). Second, 20 g sub-samples were spiked with anthracene dissolved in 2 mL acetone (anthracene-spiked soil). As such, 550 mg anthracene kg-1 was added. It was found that this concentration was sufficient to study dynamics of the contaminant in an aerobic incubation experiment. Third, 20 g sub-samples that were sterilized on three consecutive days were spiked with anthracene dissolved in 2 mL acetone under sterile conditions (anthracene-sterile soil). Fourth, 20 g sub-samples were left untreated (unamended soil). All soil samples were mixed and placed under vacuum in a desiccator for 45 min to evaporate the acetone. The aerobic incubation experiment was conducted in the same way as described in Fernández- Luqueño et al. (2008). Briefly, each soil sample (n = 2) was 5-mm sieved, adjusted to 40% WHC and incubated for 7 days. Sub-samples of 25 g from each soil sample (n = 2) were added separately to 120 ml glass flasks and the earlier mentioned treatments were applied. The glass flasks were placed separately in 1 l jars. The jars contained a 25 ml flask with 20 ml water to avoid desiccation of the soil and a 25 ml flask with 20 ml 1 M NaOH to trap evolved CO2. The soil was incubated aerobically for 56 days, while CO2 emissions, mineral N and anthracene concentrations were monitored at 0, 1, 3, 7, 14, 28 and 56 days. At the same time, a sub-sample of 5 g soil was taken and stored at -80ºC until DNA extraction. The remaining flasks were opened and aired for 10 min to maintain aerobic conditions in the soil. DOI: 10.2225/vol16-issue5-fulltext-14 2 Bacteria in anthracene-amended soil Anthracene in the soil was determined using an exhaustive ultrasonic extraction method developed by Song et al. (1995). Details of the extraction technique and the setting for the gas chromatograph (GC) can be found in Contreras-Ramos et al. (2008). DNA extraction and PCR amplification of bacterial rDNA genes The DNA was directly extracted from soil. The technique used was based on the techniques described by Valenzuela-Encinas et al. (2008). Primers 46F (5’GCC TAA CAC ATG CAA GTC 3’) and 1540R (5’GGT TAC CTT GTT ACG ACT T 3’), were used for amplification of ca. 1500 bp-long 16S rRNA gene segments from the metagenomic DNA (Edwards et al. 1989; Yu and Morrison, 2004). The PCR was done using a Touchgene Gradient thermal cycler (Techne, Cambridge United Kingdom). Cloning and sequencing PCR products The 1500 bp-long segments were used to clone and construct 16S rRNA gene libraries. The TOPO TA cloning kit with the pCR® II-TOPO® vector (Invitrogen, Carlsbad, CA) was used to clone the PCR products. Details of the cloning procedure can be found in Valenzuela-Encinas et al. (2008). The 16S rRNA gene sequences were obtained with a 3730X DNA Analyzer (Applied Biosystems, Foster City, CA) using M13 primers at the LANGEBIO (Cinvestav, Mexico). Phylogenetic and statistical analysis A total 2966 of sequences were aligned using the NAST tool from Greengenes (DeSantis et al. 2006) and chimeras were detected using Bellerophon v. 3.0 (Huber et al. 2004). The screened sequences (2898) were classified using the naïve Bayesian rRNA classifier from the Ribosomal Data Project (http://rdp.cme.msu.edu/classifier/classifier.jsp) (Wang et al. 2007). Reference sequences were obtained from the Ribosomal Database Project 10 website (http://rdp.cme.msu.edu/) using seqmatch (type and non-type strains, isolates, > 1200 bp, good quality) to construct the phylogenetic trees (Cole et al. 2009). Sequences were aligned using the NAST tool from Greengenes (DeSantis et al. 2006). Maximum likelihood phylogenetic trees were constructed with the online program PhyML 3.0 (http://www.atgc-montpellier.fr/phyml/) (Guindon et al. 2010) using the general time reversible model (GTR) (Tavaré, 1986). Rarefaction, richness and diversity indices were calculated using mothur (Schloss et al. 2009). The input files were in the form of distance matrices generated by using phylip program dnadist (Felsenstein, 1989). The Good’s coverage of our libraries was calculated (Good, 1953). Operational taxonomic units (OTUs) for community analysis were 20%, 10%, 5%, 3% distance cut-off (Rosselló- Mora and Amann, 2001). The effect of the different treatments on the different genera was analyzed by principal component analysis (PCA) using the orthogonal/varimax rotation. Data were transformed using the Hellinger transformation (Ramette, 2007).